Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates may be covalently attached to numerous other entities such as proteins and lipids, i.e., glycoconjugates. For example, the carbohydrate portion of glycoproteins may critically affect the ability of glycoproteins to perform their biological functions, including such functions as ligand or receptor recognition.
The enormous potential for chemical and structural diversity among carbohydrates is provided, in part, by the way in which individual sugar units in a polysaccharide can be linked. A fundamental step in determining the three-dimensional structure of a polysaccharide or oligosaccharide is to determine the structure of these linkages.
Many carbohydrate structures in nature are polysaccharides and oligosaccharides that are produced in a variety of related forms rather than existing in a single defined structure. These families of related carbohydrates are frequently found to be components of the same glycoconjugate. These families of glycoproteins that share the same polypeptide structure, but display variation in the glycosylation pattern have been referred to as glycoforms, Rademacher, et al., Ann. Rev. Biochem., 57:789:838 (1988). Similarly, there is great diversity in the glycoforms associated with glycolipids, proteoglycans, and polysaccharides.
The elucidation of the structure of carbohydrates and various glycoconjugates is of considerable importance in both biological and medical research. The analysis of the structures of these molecules is largely dependent on the availability of enzymes that recognize specific structures within carbohydrate molecules, i.e., the enzymes are specific for specific linkages between specific subunits of complex carbohydrates. It is thus of interest to provide enzymes that are specific for certain carbohydrate structures so as to provide for the structural analysis of newly discovered carbohydrate molecules.
It is also of interest to provide for the production of substrate-specific carbohydrate hydrolyzing enzymes by means of recombinant DNA technology. Recombinant DNA technology allows the enzymes of interest to be produced in a form that is substantially free of contaminating enzyme activity that may mask the desired activity of the enzyme of interest.
One type of carbohydrate hydrolyzing enzyme of particular interest are the neuraminidases. Neuraminidases are capable of catalyzing the release of N-acetylneuraminic acid or other forms of neuraminic acid from complex carbohydrates. Different neuraminidases are specific for the release of N-acetylneuraminic acid depending upon the specific linkage between the N-acetylneuraminic acid and the rest of the complex carbohydrate. This application describes the first isolation and discovery of a neuraminidase specific for the .alpha.2-3 linkage between N-acetylneuraminic acid and a complex carbohydrate. Also described for the first time is the recombinant DNA production of an enzyme possessing neuraminidase activity.